The etiology of human prostatic carcinoma remains largely undefined. However, it is becoming clear that epigenetic inactivation of various tumor suppressor genes could play pivotal role in the development of various cancers including prostate cancer. One of such tumor suppressor is Ras- association domain family 1 (RASSF1) gene. Epigenetic inactivation of RASSF1A is probably the most frequently methylated gene described thus far in human cancer. Ectopic expression of RASSF1A in cancer cells that lack endogenous RASSF1A transcripts resulted in reduced growth of cancer cells in vitro and in nude mice supporting a role for RASSF1A as a tumor suppressor gene. Since, the restoration of RASSF1A expression in tumor cells impairs their tumorigenicity, factors that restore RASSF1A expression have immense importance in preventing tumor growth. For long time epidemiological studies have suggested that plant derived bioactive compounds might have enormous benefit in preventing various cancers in human. In a recent study we evaluated a plant alkaloid, mahanine, isolated and purified from M. koenigii, in human prostate cancer cells and demonstrated growth inhibition in both androgen-responsive, LNCaP and androgen-independent, PC3 cells. In preliminary study, we observed that mahanine restores RASSF1A expression in human and mouse prostate cancer cells and down-regulates cyclin D1 expression. Therefore, the specific aims of this project are: 1) to determine the in vivo efficacy of mahanine in regulation of prostate cancer growth, the development of poorly-differentiated prostate cancer will be examined in TRAMP model with or without mahanine treatments. In addition, to determine the therapeutic potential of mahanine, growth inhibition of already established human prostate tumor in nude mice will be examined. Moreover, to assess mahanine is safe to use, a detailed toxicity test will be performed. 2) to determine the mechanism by which mahanine re-expresses RASSF1A in prostate cancer cells, the methylation status of RASSF1A promoter will be examined in vitro and in vivo with or without mahanine treatments. Moreover, the role of DNA methyltransferases (DNMT1, 3a and 3b) in re-expression of RASSF1A in prostate cancer cells by mahanine will be examined. 3) Finally, to determine the molecular mechanism of mahanine-induced down-regulation of cyclin D1, role of RASSF1A in inhibition of cyclin D1 will be examined. This study will demonstrate whether mahanine-induced RASSF1A regulates cyclin D1 transcriptionally via the CREB, and/or whether RASSF1A regulates cyclin D1 post-transcriptionally via the Akt. This project will provide a better understanding about the efficacy of mahanine in preventing and/or intervening prostate cancer growth in vivo and the underlying mechanisms for growth inhibition by mahanine which is crucial for future therapeutic intervention of prostate cancer in human. PUBLIC HEALTH RELEVANCE: The prostate gland is a male accessory reproductive organ and is the leading site for cancer in men over 50 years of age. According to the American Cancer Society, approximately 186,320 men will be diagnosed with prostate cancer in 2008 and annually approximately 28,600 American men are estimated to die from prostate cancer that makes prostate cancer is the most common invasive malignancy and second leading cause of cancer death in men in the United States. In the early stage of prostate cancer, the growth of prostatic cancer cells is androgen-dependent and can be effectively treated by hormone ablation either by using surgical or pharmacological castration. However, hormone ablation therapy only causes a temporary regression of prostate cancer, and tumor growth resumes within 6-18 months. Therefore better androgen blockade is not the answer for treating prostate cancer. Rather, research efforts should focus on the factor(s) that cause this deadly disease. Unfortunately, the etiology (the cause or origin of a disease or disorder) of human prostatic carcinoma remains largely unknown. However, it is becoming clear that epigenetic (reversible changes in gene function that occur without a change in the sequence of nuclear DNA) inactivation of various tumor suppressor genes could play pivotal role in the development of various cancers including prostate cancer. A large group of researchers have discovered that inactivation/silencing of the RASSF1A tumor suppressor gene is one of the most common molecular changes associated with the development of human cancer. These researchers found that RASSF1A gene is most frequently methylated (methylation is a way of inactivating genes) in human cancer, and it has been documented in at least 37 tumor types, which is surprisingly high compared to any other tumor suppressor genes we know so far. Since there is a high correlation between the inactivation/silencing of RASSF1A gene and the number of tumor types, it is possible that re-activation/restoration of RASSF1A gene in cancer cells could inhibit their growth. However, up till now, we have no idea what reactivates/re-expresses RASSF1A gene in cancer cells. Since many plant-derived bioactive compounds have anti-cancer effect, we evaluated one such bioactive compound that we isolated and purified from Indian curry leaf plant. These leaves are widely consumed in food as fresh or cooked everyday by the millions of Indian population. We demonstrated for the first time that mahanine inhibits growth in both androgen-responsive, LNCaP and androgen- independent, PC3 cancer cells in culture. In preliminary studies, we observed that mahanine re- activates/restores the epigenetically silenced tumor suppressor gene, RASSF1A in human and mouse prostate cancer cells and down-regulates cyclin D1 expression possibly thereby induces cell cycle arrest at G0/G1 phase. Therefore, the goal of this research proposal is to assess the in vivo efficacy of mahanine during the development of prostate cancer in TRAMP model, and to assess its effectiveness on already established human prostate tumor in immunodeficient mouse model in order to understand its role in therapy of prostate cancer. In addition, understanding the underlying mechanisms responsible for down- regulation of cyclin D1 in prostate cancer cells and its role in reversing epigenetically silenced tumor suppressor gene, RASSF1A in prostate cancer cells are crucial for future therapeutic intervention of prostate cancer in human.